The medullary thick ascending limb (mTAL) is critical to the regulation of salt and water homeostasis and subject to regulation via different monooxygenases that metabolize arachidonic acid. Tumor necrosis factor-alpha (TNF) production is increased in mTAL cells challenged with Angiotensin II (Ang II) and after activation of the calcium-sensing receptor (CaR), and these agonists inhibit ion transport in the mTAL in a TNF- and cyclooxygenase-2 (COX-2)-dependent manner. Thus, the contribution of TNF to prostanoid-dependent mechanisms affecting the regulation of apical K+ channels and Na+-K+-2Cl- cotransporter activity will be determined in mTAL tubules and cells, respectively. Cell will be isolated from wild type littermate control (TNF+/+) and TNF deficient mice. These mice also will be used for in vivo experiments designed to determine the contribution of TNF to the regulation of blood pressure, determined using radiotelemetry, in an Ang II-dependent model of hypertension. The ability of Ang II to regulate COX-2 expression and activity in a TNF-dependent manner also will be determined in vivo. The first demonstration that CaR activation increases nuclear factor of activated T cell (NFAT) activity in any cell type was recently provided by our laboratory. Accordingly, NFAT isoforms present in the mTAL have been identified and will be linked to the production of TNF and PGE2 in mTAL cells after CaR activation. Several cellular and molecular approaches, including electromobility shift assays, laser scanning cytometry, transfection with dominant negative NFAT constructs, and use of NFAT activity reporter constructs, will be used to identify the isoforms that contribute to CaR-mediated TNF production. The biological profile of TNF on cardiovascular function indicates that this cytokine may exhibit either pro- or anti-hypertensive activities in a context-dependent manner. For instance, in mTAL cells, TNF and its relationship to expression of COX-2 and PGE2 may act as part of an antipressor mechanism. Thus, activation of this mechanism in response to extracellular Ca2+ may be initiated by activation of CaR on the basolateral membrane of the TAL, which increases COX-2- derived PGE2 synthesis via a TNF-dependent mechanism. Our findings indicate a mechanism responsible for blood pressure reduction in response to CaR activation by virtue of the inhibitory effect of PGE2 on salt and water transport in the TAL.